1. Field of Invention
This invention relates to immunoassay procedures and it is particularly related to a method of qualitative and quantitative determination of immunological reactions. More specifically, the present invention is concerned with the application of an electro-optical imaging technique for quantification of the degree of agglutination which is present in an immunological reaction system, and the determination of the concentration of the immunoreactive components which cause the agglutination in such system. The invention is also concerned with the apparatus employed in the practice of this method.
2. The Prior Art
The assay of drugs in biological fluids has attracted considerable attention in recent years and therapeutic monitoring of drugs has achieved considerable clinical significance and has stimulated the development of new assay systems and techniques. Assay systems involving the use of antigen, haptens, or antibodies labeled with an enzyme have recently been applied to measurement of substances in various biological fluids. See G. Brian Wisdom, "Enzyme-Immunoassay," Clinical Chemistry, Vol. 22, No. 8 (1976), pp. 1243-55. These assay systems are known as enzyme immunoassay, generally abbreviated as "EIA" and enzyme-linked immunoassay, abbreviated as "ELISA." Other techniques include radio-immunoassay (RIA) which involves producing hapten protein conjugates, and radiolabelling at specific sites on the drug molecule. See Alan Broughton and James E. Strong, "Radioimmunoassay of Antibiotics and Chemotherapeutic Agents, Clinical Chemistry, Vol. 22, No. 6 (1976), pp. 726-32, and R. Cleeland et al, "Detection of Drugs of Abuse by Radio Immunoassay: A Summary of Published Data and Some New Information." Clinical Chemistry, Vol. 22, No. 6 (1976), pp. 713-25.
Prior to the development of the various recent assay techniques, the presence of antigen/antibody complexes were usually detected visually by the naked eye. However, the naked eye could not detect or differentiate marginal, but clinically meaningful reactions and, therefore, the results often had limited clinical significance. Even microscopic examination afforded limited clinical information since it could not provide quantitative results.
While EIA techniques, in general, provide specific and highly sensitive methods for identification and quantification of wide ranges of substances, they suffer from several disadvantages and limitations. These techniques are generally less sensitive than RIA and they are more susceptible to interference. Additionally, the determination of end point (the initial velocity of enzymatic reaction) is more difficult than in RIA techniques, and, moreover all EIA techniques, except homogeneous EIA, require separation of the bound molecules from the free labeled molecules. However, the number of separation methods which can be applied are usually limited.
The RIA techniques, on the other hand, require the production of a specific antibody and the development of a suitable radioactive labeled compound. The application of this technique requires extreme care and expertise due to hazard of radiation and the radiation damage may even adversely affect the immunochemical reactivity of the labelled substance.
Other known assay systems include spin-immunoassay in which known amounts of antibodies to the drug to be detected are mixed with an analog of the drug that has been labeled with nitroxide label (spin label) and the specimen of biological fluid is then added to this mixture. The drug concentration in the unknown sample is then determined by electron spin measurement. See Simon L. Sharpe, "Quantitative Enzyme Immunoassay: Current Status," Clinical Chemistry, Vo. 22, No. 6 (1976), pp. 733-38.
Still other assay techniques are available which include the attachment of antibodies to fluorescent compounds capable of emitting light when excited by illumination of specific wavelengths; and the utilization of opaque, colloidal particles such as latex spheres, glass and ceramic spheres, kaolin, carbon and charcoal particles, as well as animal blood components, typically erythrocytes, and attaching the antigen/antibody thereto.
In the aforementioned immunoassay techniques, the labeled component of an antigen/antibody reaction binds to its complementary site, and the amount bound depends upon the concentration of the other component, and if one of these concentrations is varied, there will be a concomittant change in the distribution of the labelled component between the bound and unbound fractions. The properties of the labels determine its distribution and a calibration curve can be constructed relating the concentration of the variable (unlabeled) component to the labeled component. Presently, this is accomplished by the removal of either the bound or unbound fraction by means of solid-phase absorbtion in test tubes or plastic beads, centrifugation, repeated resuspension and washing steps. Although, these procedures provide a curve defining the relationship between the bound and unbound fractions, they are tedious, cumbersome and require complicated equipment. Additionally, EIA, RIA and spin immunoassay usually require the introduction into the reactive system labels which, while not immunoreactive, they may adversely affect the immunoreactivity of the system through the chemical or radioemissive activity which permits their use as the labels.
The classical visual indicators of immunoreactivity do not suffer from the disadvantages of EIA, RIA and spin immunoassay procedures since they do not require phase separation and removal of the components from the immunoreactive system, nor do they require the introduction of labels which interfere with or modify the immunoreactivity of the system under consideration. Rather, they are based on redistribution of the opaque particles from a uniform non-agglutinated state to an agglutinated state. Since each particle serves as the label for numerous immunoreactive protein molecules, a number of labeled particles are agglutinated to form a characteristic microscopic agglutination texture, which is read qualitatively by visual inspection. However, this procedure does not lend itself to quantitative measurement and hence it is likewise of very limited clinical significance.
There are also numerous patents which relate to different methods and approaches for the detection of agglutination reactions. Thus, U.S. Pat. No. 3,074,853 (Brewer) discloses a method and means for carrying out immunological reactions by forming and spreading a mixture of a finely divided solid and two liquids to be tested for antigen/antibody reaction in a test spot on an opaque surface of contrasting color with respect to said solid. The test spot is then examined visually.
U.S. Pat. No. 3,520,609 (Lion) discloses a method for detecting agglutination reactions which comprises scanning samples of the specimen by a beam of energy (e.g., light or electrons). Substantial agglutination establishes significant demarcations in the scanned zone between the agglutinated cells and the surrounding area, and these demarcations modulate the scanning beam and it crosses them, causing a change in rate in the signal produced thereby. A second signal is derived from the rate of change of the signal generated by the beam of light, integrated a predetermined period and the integrated value is compared with a predetermined standard, corresponding to substantial agglutination to determine if such agglutination has occurred in the reaction area.
U.S. Pat. No. 3,819,271 (Beug) describes a method and an apparatus for the measurement of cell agglutination in a carrier liquid. According to the procedure described in this patent, a carrier liquid having cells suspended therein is enclosed in a container which is moved in a circular path and a beam of light is passed through the suspension. The degree of agglutination of the cells is determined from the amount of light "scattered" by passage through the suspension.
U.S. Pat. No. 3,984,533 (Uzgiris) describes an electrophoretic method of detecting antigen/antibody reactions, and U.S. Pat. No. 3,990,851 (Gross et al) describes a process and a device for measuring antigen/antibody reactions by passing a laser light through the reaction mixture and measuring the light "scattered" in the forward direction.
U.S. Pat. No. 3,905,767 (Morris et al) describes a process for qualitative or quantitative measurement of antigen or antibodies with an antigen/antibody reaction in which a "precipitate" is formed. This patent also relies on measuring the extent of light "scattered" by the precipitate when a light beam is projected therethrough.
The methods and apparatus described in the aforementioned patents, and other prior art patents, all require detection of the agglutination reaction by photometric means such as measurement of light scatter, or turbidimetry or opacimetry. Consequently, the measurements are subject to clinically significant errors due to the color or inherent cloudiness of the biological fluid under examination. Moreover, the procedure described in all the aforementioned patents, save for Beug, supra, suffer from uneven distribution of reactant indicators which causes statistical errors upon inspection by automatic means and difficulty in interpretation by manual techniques.
Thus, there is a long-felt need for an assay procedure for the detection and quantification of immunological reactions which is simpler and more rapid to carry out, and which provides clinically more significant information without the difficulties and limitations which are inherent in, or associated with, the currently available immunoassay techniques.
It is therefore an object of this invention to provide an immunoassay procedure, and an apparatus for carrying out the same, for the detection and quantification of immunological reactions.
It is a further object of this invention to provide an immunoassay procedure which provides clinically more significant information as compared with the currently available immunoassay techniques and systems.
It is still a further object of this invention to provide an immunoassay system and procedure in which the detection and quantification of immunological reactions may be carried out more rapidly and more accurately than the prior art systems and procedures.
It is yet another object of this invention to provide an immunoassay procedure which is non-invasive and which does not require separation of the bound fraction from the unbound fraction.
It is still a further object of this invention to provide an immunoassay technique and system capable of extracting a greater proportion of the information available from the labeled components.
It is also an object of this invention to provide a uniquely designed, disposable, planar reaction imaging cell in which the immunological reaction is carried out.
It is a further object of this invention to provide a holder for securing the reaction imaging cell in the apparatus of this invention during image analysis of the reactants in the image cell.
The foregoing and other objects of this invention will become more apparent from the ensuing detailed description taken in conjunction with the accompanying drawings, wherein like reference numerals are employed to designate like parts.